Retroviruses are major causes of disease in animals and humans. Retroviruses replicate by reverse transcribing viral RNA into DNA, using the virus-encoded transcriptase. Since uncoating as well as reverse transcription occur in the cytoplasm, there is the potential for recognition of ?foreign? viral RNA or DNA by host sensors. Recently, a number of host sensors, including cGAS, DDX41 and the ALR IFI16, have been implicated in the recognition of cytosolic DNA. Using a mutant murine leukemia virus (MLV) with an unstable capsid that induces a strong IFN? response, we found that reverse transcripts induced this response and identified three sensors in mice required for recognition ? IFI203, DDX41 and cGAS - that signal via the STING pathway leading to increased IFN?? Using APOBEC3 knockout and STING mutant mice and cells, we showed that the host retroviral restriction factor APOBEC3 limits the levels of reverse transcripts that trigger cytosolic sensing. Moreover, we found that the role of nucleic acid sensing in vivo is to increased expression of IFN- regulated restriction factors like APOBEC3 that in turn reduce viral load. While the identification of sensors involved in recognition is an important first step, there as of yet many unanswered questions. While we and others have shown that host sensing of retroviral nucleic acid is dependent on reverse transcription and therefore must include DNA detection, the involvement of at least 3 different factors in the response to infection could mean that RNA or RNA/DNA are also recognized. Additionally, while it is well-accepted that DNA binding cGAS activates production of cyclic GMP-AMP and that this ligand in turn activates STING, whether IFI203 and DDX41 operate in the same or parallel pathways to induce IFN is not known. Finally, the relative importance of the different host sensors in controlling viral infection in vivo has yet to be elucidated. To address these questions, we propose to carry out the following aims: I. What retroviral nucleic acids serve as ligands for cGAS, IFI203 and DDX41? II. What role does each of the sensors play in in vivo control of infection? III. What is the pathway of action of cGAS, IFI203 and DDX41 in the response to retroviral infection? Understanding the initial host response to infection by retroviruses is critical to our ability to determine how these viruses establish persistent infection as well the discovery of novel approaches to intervene in these infections. Using a combination of functional and genetic approaches, this proposal will delineate the molecular means by which retroviral nucleic acids are sensed by cells, as well as to determine the significance of this sensing in in vivo infection and pathogenesis.